PROJECT SUMMARY Mechanical forces play fundamental roles in many intrinsic and collective cellular processes, including tissue regeneration, morphogenesis, and tumor metastasis. While extensive studies have focused on the forces between cells and extracellular matrices, mechanical interactions among individual cells appear to be important yet poorly characterized. These intercellular forces are known to be critical during wound healing, cancer cell invasion, and other developmental and homeostatic processes. However, the molecular principles that govern these finely balanced mechanotransduction events are still poorly understood. To depict the mechanisms of these collective cellular processes, it is essential to measure intercellular forces and correlate the determined mechanical landscapes with the specific molecular machineries that regulate cellular signaling. Our lab have proposed precise and easy-to-use DNA-based sensors to visualize and quantify intercellular forces. We and others recently developed an efficient lipid-based approach to anchor designer DNA sequences onto the external surfaces of mammalian cell membranes. By employing this approach, membrane-anchored DNA probes allow sensitive imaging of a broad range of molecular forces at cell-cell junctions. Current mechanobiology studies are based on techniques typically performed in only a few specialized laboratories. The proposed sensors are compatible with readily accessible fluorescence microscopes, highly robust and versatile, and easy to prepare and use. To further develop and adapt these sensors to study intercellular mechanosensitive events, the future research plan is to: (1) engineer and optimize DNA-based tensile and compressive force sensors to measure a broad range of intercellular forces at the single-molecule level. (2) Use well-characterized cadherin-based mechanotransduction as an example, quantify and monitor forces at cell-cell junctions during collective cell migrations and neural plate shaping. (3) Apply these sensors to investigate the mechanical roles of Notch activation in immune cell activation. Notch signaling is highly conserved in different developmental and disease processes. Intercellular ligand-induced mechanical forces are required in Notch activation. Dependent on the environmental and mechanical context, Notch activation can have contrary effect in the regulation of tissue growth and immune responses. Our results will provide unique insights to elucidate the mechanical mechanisms of Notch signal activation. Our long-term goal is to make intercellular force measurements widely implemented in life science laboratories. These novel sensors will be broadly used to understand the basic mechanical principles of development, physiology, and disease, which will also serve as the critical foundation for developing novel strategies in tissue engineering, regenerative medicine, and cell therapy.